Flavonoids Biotransformation by Human Gut Bacterium Dorea sp. MRG-IFC3 Cell-Free Extract

Human gut bacterium Dorea sp. MRG-IFC3 is unique in that it is capable of metabolizing puerarin, an isoflavone C-glycoside, whereas it shows broad substrate glycosidase activity for the various flavonoid O-glycosides. To address the question on the substrate specificity, as well as biochemical characteristics, cell-free biotransformation of flavonoid glycosides was performed under various conditions. The results showed that there are two different enzyme systems responsible for the metabolism of flavonoid C-glycosides and O-glycosides in the MRG-IFC3 strain. The system responsible for the conversion of puerarin was inducible and comprised of two enzymes. One enzyme oxidizes puerarin to 3”-oxo-puerarin and the other enzyme converts 3”-oxo-puearin to daidzein. The second enzyme was only active toward 3”-oxo-puerarin. The activity of puerarin conversion to daidzein was enhanced in the presence of Mn2+ and NAD+. It was concluded that the puerarin C-deglycosylation by Dorea sp. MRG-IFC3 possibly adopts the same biochemical mechanism as the strain PUE, a species of Dorea longicatena.


Activity of Cell-Free Extract
All the experimental procedures, including screening, isolation, and identification of bacteria, were performed under anaerobic conditions (CO 2 5%, H 2 10%, N 2 85%) at 37°C, except HPLC analysis.For the cell-free extract, Dorea sp.MRG-IFC3 was cultured under anaerobic conditions at 37 o C for 20 h in 1 L of GAM broth containing 128.8 mg (0.3 mM) puerarin as an enzyme inducer.The bacteria cells were collected by centrifugation (7000 ×g, 10 min) when the OD 600 reached 2.2.The harvested pellet was washed with 50 mM phosphate buffer (pH 7.4, 50 ml) before storage in liquid nitrogen.For the cell lysis, cell pellet (2 g) was resuspended in 10 ml of phosphate buffer (50 mM, pH 7.4), and disrupted by sonication at 60% amplitude for 30 min with a cycle of 10 sec on and 20 sec off at 0°C.The cell lysate was then centrifuged at 13,000 g for 90 min at 4 o C to obtain the supernatant as a cell-free extract.The cell-free extract was also prepared under argon atmosphere to test the air-sensitivity.In detail, degassed buffers were prepared by vacuum evacuation with sonication, and centrifugation was performed under argon.The reaction was performed in the anaerobic chamber.The cells without puerarin inducer were processed by the same method, except that the culture broth did not contain puerarin.
To check the activity of cell-free extract, 0.2 mM of each substrate was reacted at 37°C.Aliquots of the reaction (100 μl) were taken, allocated into microcentrifuge tubes, and extracted with 1 ml ethyl acetate.Then, 800 μl supernatant, collected after vortexing and centrifuging at 10,770 g for 10 min, was dried under vacuum.The dried residue was dissolved in 100 μl MeOH, and filtered through a 0.2 μm PTFE syringe filter (Advantec, Japan) for the analysis by HPLC.
HPLC analysis was conducted by UHPLC-DAD (Thermo Fisher Scientific, USA), with a kinetex C18 column (1.7 mm particle size; 100 × 2.1 mm i.d., USA) at 35 o C. The flow rate was 0.2 ml/min with the mobile phase consisting of 0.1% formic acid (v/v) in water (A) and acetonitrile (B).The eluent started with solvent B from 5 to 55% in 20 min and was held at 55% for 5 min, then increased linearly to 100% in 5 min and maintained for 3 min.After the analysis, the composition of the eluent was returned to 5% B in 2 min linearly.The injection volume was 1.0 ml.Program setup, data collection, and analysis were performed using Chromelon Chromatography Data System software version 6.8 (Thermo Fisher Scientific).

Cell-Free Extract Reaction in the Presence of Additives
Cell-free extract was incubated with 0.2 mM of each substrate (puerarin or daidzin) in 50 mM potassium phosphate buffer (pH 7.4) containing 1 mM Mn 2+ and 1 mM NAD + at 37ºC.Aliquots of the reaction mixture (100 μl) were taken, allocated into microcentrifuge tubes, and extracted with 1 ml ethyl acetate.Then, 800 μl supernatant was collected after vortexing and centrifuging at 10,770 g for 10 min, followed by drying under vacuum.The dried residue was dissolved in 100 μl MeOH filtered through a 0.2 μm PTFE filter (Advantec, Japan) for the chromatography analysis by HPLC.

Results and Discussion
Currently, three gene clusters, dfg, dgp, and car from Eubacterium cellulosolvens, Dorea strain PUE and Microbacterium strain 5-2b, respectively, are reported to exhibit C-deglycosylation activity [6,13,14].However, DgpA and DgpBC from dgp operon are the only enzymes of which the biochemical properties were investigated at the molecular level.Thus, our understanding of biochemical C-deglycosylation is limited to the mechanism proposed from the strain PUE, so that it is not certain whether other natural C-glycosides would follow the same biochemical mechanism.In this report, the results obtained from flavonoid biotransformation by the cell-free extract of Dorea sp.MRG-IFC3 have provided a number of significant findings.

Puerarin C-Deglycosidase from Dorea sp. MRG-IFC3 is Inducible
Dorea sp.MRG-IFC3 is a strict anaerobe.First, air-sensitivity of C-deglycosylation was tested by comparing the reactivity of cell-free extracts in the presence and absence of air.The activity of conversion of C/O-glycosides, such as puerarin and daidzin, was not affected by air.Therefore, it was determined that glycoside metabolizing activity does not require the air-sensitive cofactors, such as Fe/S cluster.Secondly, inducibility of C-deglycosylation was investigated by comparing the puerarin conversion activity of the cell-free extracts prepared from the cells grown in the presence and absence of puerarin.The cell-free extract prepared from the cells grown with 0.3 mM puerarin

Fig. 2. HPLC chromatograms of C-glycosides reaction products of the induced cell-free extract.
After 2 h of reaction time, puerarin (A) was converted to two minor products (peak 1 and 2) as well as daidzein.Vitexin (B) was converted to two minor products (peak 3 and 4), orientin (C) was converted to two minor products (peak 5 and 6), and isoorientin (D) was converted to two minor products (peak 7 and 8).completely converted puerarin in 60 min (Fig. 1A), whereas that from the cell grown without puerarin did not show any activity at all (Fig. 1B).Therefore, it was concluded that the enzyme responsible for puerarin conversion to daidzein is inducible.
Furthermore, it was found that the enzyme responsible for O-glycoside conversion is different from the Cdeglycosidase.Both cell-free extracts, prepared from the cells grown in the presence and absence of puerarin, exhibited the same O-glycoside conversion activity.When time-dependent C/O-glycoside conversions by the cellfree extract were compared, the conversion of C-glycosides puerarin was slower than that of O-glycoside daidzin (Table 1).Besides, puerarin conversion was not completed even after depletion of daidzin.The results confirmed that the activities of C/Oglycosidic bond cleavage by Dorea sp.MRG-IFC3 are achieved by different enzymes and the enzyme responsible for the C-glycoside metabolism is inducible.

MRG-IFC3 only Converts Isoflavone C-Glycoside Puerarin
As reported previously from whole cell biotransformation, Dorea sp.MRG-IFC3 metabolized only puerarin, an isoflavone C-glycoside.It does not metabolize flavone C-glycosides, such as vitexin, orientin and isoorientin [12].One of the possible explanations was the cell transporter which is specific to puerarin, similar to the cell signaling during the nodule formation of the symbiotic nitrogen-fixing soil bacteria [15].When we measured the reactivity of cell-free extract with various C-glycosides, no difference has been observed, except reaction rate of puerarin conversion (Fig. 2).Therefore, it was concluded that the high substrate specificity by Dorea sp.MRG-IFC3 Cdeglycosidase toward C-glycosides is an intrinsic property.
On the other hand, all the O-glycosides, including flavone O-glycosides and isoflavone O-glycosides, were converted by the cell-free extract and the aglycones were produced.No other possible peaks for byproducts or intermediates were observed from the reaction of O-glycosides (Table 1).

Puerarin C-deglycosidase from Dorea sp. MRG-IFC3 May Adopt the Same Mechanism as C-Deglycosidase of the Strain PUE
Previously, no intermediate was observed during the whole cell biotransformation of C-glycosides [12].Interestingly, two minor metabolites were observed from the cell-free extract biotransformation of C-glycosides (Fig. 2).These two metabolites, detected right after the substrates on the HPLC chromatograms, were identified as 3"-and 2"-oxo-products by LC-MS analysis, as reported by others [6,13,16].Except puerarin (Fig. 2A), no aglycone productions for the other three C-glycosides were observed (Fig. 2B-2D).On the contrary, the oxoproducts were not observed from O-glycosides reactions by cell-free extract, which again confirmed that C-/Oglycosides metabolism by Dorea sp.MRG-IFC3 are performed by different enzyme systems.
The activity of puerarin biotransformation by the cell-free extract was influenced by the additives, Mn 2+ and NAD + and both significantly increased the daidzein production (Fig. 3).NAD + was proposed as a cofactor of DgpA glycoside oxidoreductase and Mn 2+ as a cofactor of DgpBC C-deglycosidase from the strain PUE [7,8].Besides, it is noteworthy that the amounts of 3"-oxo-puerarin was significantly reduced in the presence of Mn 2+ (Fig. 3B).Therefore, it is proposed that C-deglycosylation of Dorea sp.MRG-IFC3 follows the same reaction mechanism as the strain PUE.Though, it is still not clear why it does not react with other C-glycosides.Based on our observation shown at Fig. 2, it is clear that the other C-glycosides, such as vitexin, orientin, and isoorientin, were converted to the oxo-intermediates.Hence, we propose that the substrate binding site of C-deglycosidase of Dorea sp.MRG-IFC3 is different from that of the PUE strain [7].

Conclusion
Biochemical C-deglycosylation is known to occur in two steps [7].Namely, puerarin needs to be oxidized to 3"oxo-puerarin, before the cleavage reaction of the glycosidic C-C bond.The former reaction was proposed being catalyzed by NAD + -dependent oxidoreductase DgpA, and the latter performed by Mn 2+ ion-dependent C-

Fig. 1 .
Fig. 1.Puerarin biotransformation by cell-free extracts of Dorea sp.MRG-IFC grown in the presence (A) and in the absence (B) of puerarin.The reaction time was 60 min.

Fig. 3 .
Fig. 3. HPLC chromatograms of puerarin reaction products by the induced cell-free extracts of Dorea sp.MRG-IFC3 in the presence of NAD + and/or Mn 2+ .Daidzin production from puerarin was highest in the presence of NAD + and Mn 2+ (A) compared to the reactions with only Mn 2+ (B) or only NAD + (C).The reaction without any cofactors (D) was performed as control experiment.